1. Field of the Invention (Technical Field):
This invention relates to a method and composition for labeling proteins, peptides and amino acids, including antibodies, with a variety of metals ions, including radioisotopes of a variety of radiometals.
2. Background Art:
The use of radioisotopes to label proteins is well known. These compositions can be used in in vitro assays; can be administered to the human body to visualize or monitor functioning of various parts of the body or to determine the presence and location of particular antigens, antibodies, hormones or the like; and can be used in the treatment of various disease states. A variety of radioisotopes, including isotopes of technetium, indium, copper, rhenium, gold and arsenic have been used to label proteins.
Antibodies and antibody fragments have been labeled with a number of radionuclides for use in clinical diagnosis. These radionuclides include .sup.131 I, .sup.125 I, .sup.123 I, .sup.99m Tc, .sup.67 Ga, and .sup.111 In. So far, only .sup.99m Tc and .sup.111 In-labeled antibody preparations are widely used in clinical settings. For diagnostic imaging, both isotopes should be ideal; however, clinical limitations, including affinities for liver and kidneys that limit detection of abdominal diseases, have prompted searches for other imaging radionuclides.
Antibodies have also been labeled with a variety of radionuclides for potential use in targeted immunotherapy (Pietersz G A, Kannellos J, Smyth M J, et al. The use of monoclonal antibody conjugates for the diagnosis and treatment of cancer. Immunol Cell Biol 65:111-125, 1987). These radionuclides include .sup.90 Y, .sup.188 Re, and .sup.186 Re, and to a lesser extent .sup.199 Au and .sup.67 Cu. .sup.131 I has also been used. With the exception of .sup.131 I, all the methods currently used to conjugate these radiometals to antibodies involve the use of chelating groups chemically attached to the antibody. .sup.67 Cu is one radionuclide that has been specifically recommended for use as a therapeutic radionuclide when bound to antibodies (DeNardo G L, Raventos A, Hines H H, et al. Requirements for a treatment planning system for radioimmunotherapy. Int J Radiol Oncology Biol Phys 11:335-348, 1985). .sup.199 Au-conjugated monoclonal antibodies have also been suggested for potential use as cancer therapeutic agents.
.sup.67 Cu has been attached to monoclonal antibodies through chelates, e.g., a macrocycle chelate (6-para-nitrobenzyl-1,4,8,11-tetraazacyclotetradecane-N,N',N",N"') (Deshpande S V, DeNardo S J, Meares C F, et al. Copper-67-labeled monoclonal antibody Lym-1, A potential radiopharmaceutical for cancer therapy: labeling and biodistribution in RAJI tumored mice, J Nucl Med 29:217-225, 1988), and porphyrins (Roberts J C, Figard S D, Mercer-Smith J A, et al. Preparation and characterization of copper-67 porphyrin-antibody conjugates. J Immunol Meth 105:153-164, 1987). The macrocycle chelate, but not the porphyrin conjugate, was evaluated in an animal model system. Both .sup.64 Cu and .sup.67 Cu, have been conjugated by the porphyrin method to antibodies and autoantigenic peptides (Roberts J C, Newmyer S L, Mercer-Smith J A, Schrerer and Lavallee D K. Labelling antibodies with copper radionuclides using N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine. Appl Radiat Isot 40:775-781, 1989). Biodistribution studies of radiocopper-labeled antibodies have shown that blood clearance is rapid and uptake to the bone is low (Mercer-Smith J A, Cole D A, Roberts J C, et al. The biodistribution of radiocopper-labeled compounds. In: C Kies (ed), Copper Bioavailability and Metabolism, pp 103-121, 1990).
Antibodies have been labeled with .sup.199 Au, in the form of gold clusters (Hainseld J F, Foley C J, Srivastava S C, et al. Radioactive gold cluster immunoconjugates: Potential agents for cancer therapy. Nucl Med Biol 17:287-294, 1990), and with .sup.199 Au and .sup.195 Au, as complex ions in citrate buffered saline (Anderson P, Vaugan A T M, and Varley N R. Antibodies labeled with .sup.199 Au: Potential use of .sup.199 Au for radioimmunotherapy. Nucl Med Biol 15:293-297, 1988).
Antibodies and other proteins have been directly labeled. Although several direct methods have been reported, the first direct method capable of providing a sufficiently strong bond between the protein and technetium-99m for In vivo applications was the direct or pretinning method described in U.S. Pat. No. 4,424,200, entitled Method for Radiolabeling Proteins with Technetium-99m, to Crockford, D. R., and Rhodes, B. A. In this method, a single reduction compound, consisting of stannous Sn(II)! chloride and other salts which serves both to reduce the protein, thereby exposing the disulfide bonds, and to reduce the sodium pertechnetate, is used. With this method, many proteins can be successfully radiolabeled with .sup.99m Tc. Several investigators have reported on the use of this method (Rhodes, B. A., et al, "Technetium-99m labeling of murine monoclonal antibody fragments," J Nucl Med 27:685-693, 1986; Som, P., et al, "Radioimmunoimaging of experimental thrombi in dogs using technetium-99m-labeled monoclonal antibody fragments reactive with human platelets," J Nucl Med 27:1315-1320, 1987).
Equivalent methods for direct labeling have been reported (Schwarz, A., and Steinstruaber, A., "A novel approach to Tc-99m-labeled monoclonal antibodies," J Nucl Med 28:721, 1987; Pak, K. Y., et al, "A rapid and efficient method for labeling IgG antibodies with Tc-99m and comparison to Tc-99m Fab'". J Nucl Med 30:793, 1989; Granowska, M., et al, "A Tc-99m-labeled monoclonal antibody, PR1A3, for radioimmunoscintigraphy," J Nucl Med 30:748, 1989). In the equivalent methods disulfide reducing agents other than stannous salts were used. Pak et al used dithiothreitol to reduce the disulfide bonds of the antibody; Swartz and Steinstrauber, and Granowska et al used 2-mercaptoethanol. Also some of these investigators (Swartz and Steinsbruaber, and Granowska et al) reduced the Tc-99m prior to adding it to the reduced antibody, which adds steps to the original procedure.
Reno, J. W., et al, U.S. Pat. No. 4,877,868, Radionuclide Antibody Coupling, uses dithiothreitol (DTT) to reduce the disulfide groups of the protein, then protects the reactive sulfides with Zn (II) or other sulfhydryl group derivatizing reagents. Tartrate salts are used to complex and transfer the reduced radionuclide. This method uses potentially toxic chemicals, such as dithiothreitol, to reduce the antibody. It also requires multiple steps to radiolabel the protein.
Thakur, M. L., U.S. Pat. No. 5,011,676, Method to Directly Radiolabel Antibodies for Diagnostic Imaging and Therapy, used sodium ascorbate to reduce the disulfide groups of antibodies. However, this method cannot be adapted to single-step, direct labeling; it is required to reduce the radionuclide prior to adding the radionuclide to the sodium ascorbate reduced protein. In a preferred embodiment of the Thakur method, a separate vial is utilized, in which sodium dithionite is used to reduce the radionuclide, producing dithionite reduced radionuclide.
There are useful metals for magnetic resonance imaging, including gadolinium, manganese, copper, iron, gold and europium, which are not radioisotopes. Examples also include ions of a lanthinide element of atomic numbers 57-70 or ions of transition metals of atomic numbers 21-29 and 42-44. Examples of metals which would be expected to be of potential utility in magnetic resonance imaging with proteins labeled by the methods described in the present invention include copper, iron and gold, as well as colloidal preparations of iron or gold.
So far, antibodies do not appear to have been labeled with positron-emitting radiometals, although other types of proteins (transferrin and human serum albumin) have been labeled with .sup.68 Ga (Green M A, and Welch M J. Gallium radiopharmaceutical chemistry. Nucl Med Biol 16:435-448, 1989). The short half-life associated with .sup.68 Ga, i.e., 68 minutes, suggests that it often may not be a suitable label for targeting antibodies, which tend to have prolonged biological half-lives.